Systematic characterisation of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry
- Division of Molecular, Cell and Developmental Biology, Faculty of Life Sciences, University of Dundee, United Kingdom
- Institute of Systems, Molecular and Integrative Biology, University of Liverpool, United Kingdom
- Bioscience Core Lab, King Abdullah University of Science and Technology, Saudi Arabia
Figures
Figure 1
Utilizing Hydrophilic Interaction Liquid Chromatography (HILIC) for the enrichment of hydroxylated proline peptides.
(A) The schematic diagram of proline hydroxylation. (B) Hydrophilic interaction liquid chromatography (HILIC) showing the retention time difference of the synthetic peptides (HLSSLAPP, with no modification, or hydroxylation at the histidine/proline). (C) HILIC profile of peptides fractionation from RCC4 samples (3 replicates, overlay).
Figure 2 with 1 supplement
HEK293 dataset.
(A) The hydrophilicity difference of peptides with hydroxylation (P), oxidation (M), and unmodified peptides across hydrophilic interaction liquid chromatography (HILIC) fractions from all experiments with different treatments. To generate the heatmap, ‘modificationSpecificpeptides.txt’ result file was used and divided into three files for identified peptides with either hydroxylation (P), oxidation (M), or unmodified peptides. The value of ‘Fraction x’ column from each peptide was used and summarised for each fraction, which represented the total quantity of peptides in each fraction. In each condition, the summarised fraction value of F1, F2, …, F24 was scaled to 100% in total. The scaled fraction value was used for heatmap analysis by Heatmapper2 (Nucleic Acids Res. 2025 May 05. doi:10.1093/nar/gkaf385). The hydrophilicity increases from Fraction 1 to Fraction 32 (XF1…XF32). (B) The charge distribution of peptides with hydroxylation (P), oxidation (M), and unmodified peptides. (C) The mass distribution of peptides with hydroxylation (P), oxidation (M), and unmodified peptides. (D) The missed cleavage difference of peptides with hydroxylation (P), oxidation (M), and unmodified peptides.
Figure 2—figure supplement 1
RCC4 dataset.
(A) The hydrophilicity difference of peptides with hydroxylation (P), oxidation (M), and unmodified peptides across hydrophilic interaction liquid chromatography (HILIC) fractions from all experiments with different treatments. To generate the heatmap, ‘modificationSpecificpeptides.txt’ result file was used and divided into three files for identified peptides with either hydroxylation (P), oxidation (M), or unmodified peptides. The value of ‘Fraction x’ column from each peptide was used and summarised for each fraction, which represented the total quantity of peptides in each fraction. In each condition, the summarised fraction value of F1, F2, …, F24 was scaled to 100% in total. The scaled fraction value was used for heatmap analysis by Heatmapper2 (Nucleic Acids Res. 2025 May 05. doi:10.1093/nar/gkaf385). The hydrophilicity increases from Fraction 1 to Fraction 32 (XF1…XF32). (B) The charge distribution of peptides with hydroxylation (P), oxidation (M), and unmodified peptides. (C) The mass distribution of peptides with hydroxylation (P), oxidation (M), and unmodified peptides. (D) The missed cleavage difference of peptides with hydroxylation (P), oxidation (M), and unmodified peptides.
Figure 3 with 1 supplement
The MS intensity behaviour of HyPro diagnostic ion using synthetic peptides.
(A) The intensity behaviour of HyPro diagnostic ion (m/z at 86.060) under different loading amount (left) and NCE settings (right), using the synthetic peptide HIF1α-P564, LDLEMLAPYIPMDDD (m/z at 833.9018). (B) The intensity behaviour of HyPro diagnostic ion (m/z at 86.060) under different loading amount (left) and NCE settings (right), using the synthetic peptide CEP192–1717, WHLSSLAPPYVK (m/z at 707.38). (C) The intensity behaviour of HyPro diagnostic ion (m/z at 86.060) under different loading amount (left) and NCE settings (right), using the synthetic peptide LAPITSDP (8) TEATAVGAVEASFK (m/z at 707.38). All ions were extracted under ±10 ppm mass tolerance.
Figure 3—figure supplement 1
The structure, mass, and immonium ion difference of leucine, isoleucine, and hydroxyproline.
Figure 4 with 1 supplement
Sequence logo analysis for identified hydroxylated proline sites in HEK293 dataset.
(A) Sequence logo for peptide window with hydroxylated proline site from collagen proteins (with diagnostic ion) and from other proteins (with diagnostic ion). (B) Sequence logo for peptide window with hydroxylated proline site from all the proteins. (C) Sequence logo for proline-hydroxylated peptides from proteins included in different protein families (RRM_1 domain, pkinase domain, Helicase_C domain, WD40 domain, and DEAD domain).
Figure 4—figure supplement 1
The percentage of M in total amino acids at each position from (A) HEK293 and (B) RCC4 dataset, centred at the hydroxyproline residue.
Figure 5
Sequence logo analysis for identified hydroxylated proline sites in RCC4 dataset.
(A) Sequence logo for peptide window with hydroxylated proline site from collagen proteins (with diagnostic ion) and from other proteins (with diagnostic ion). (B) Sequence logo for peptide window with hydroxylated proline site from all the proteins. (C) Sequence logo for proline-hydroxylated peptides from proteins included in different protein families (RRM_1 domain, pkinase domain, CH domain, WD40 domain, and LIM domain).
Figure 6
Pathway analysis of proteins with proline hydroxylation sites in the FG-4592 dependent dataset.
(A) Statistical gene enrichment pathway analysis (Reactome) of the proteins only hydroxylated in DMSO/MG-132-treated HEK293 cells but not in FG-4592 treatment. (B) Statistical gene enrichment pathway analysis (Reactome) of the proteins only hydroxylated in DMSO/MG-132-treated RCC4 cells but not in FG-4592 treatment. (C) The Venn diagram of the identified proline hydroxylation sites between HEK293 and RCC4 dataset. (D) Statistical gene enrichment pathway analysis (Reactome) of the proteins, which not only have the same hydroxylation proline sites from HEK293 and RCC4 dataset (DMSO/MG-132-treated), but also not hydroxylated in any FG-4592 treatment. (E) RCC4 cells were treated with the indicated siRNAs, and the cell cycle profile was determined by flow cytometry. The values presented represent the averages from three independent experiments. The error bars indicate the standard deviations (n=3). (F) RCC4 cells were treated with the PHD inhibitor FG-4592 for the indicated times points prior to fixing. After which the cell cycle profile was determined by flow cytometry. The averages from three independent experiments are presented, whilst the error bars indicate the standard deviations (n=3). In both (E) and (F), the cell profiles were gated according to the control in each independent experiment, and the number of cells in each phase are expressed as a percentage of the total number of cells present. Statistical analysis was performed according to the Student’s t test vs the control; *p<0.05.
Figure 7
The confirmation of P604 hydorxylation in CDCA2 using synthetic peptides.
(A) The retention time difference of the synthetic peptides (KPLLSPIPELPEVPEMTPSIPSIRR) with or without oxidation/hydroxylation (+16 Da mass shift) at different amino acids using RPLC-MS analysis, individually. (B) The retention time difference of the synthetic peptides (KPLLSPIPELPEVPEMTPSIPSIRR, K+8 Da) with or without oxidation/hydroxylation (+16 Da mass shift) at different amino acids using RPLC-MS analysis, mixed. (C) The retention time comparison of the M602 oxidised peptides (KPLLSPIPELPEVPEM(O)TPSIPSIRR) with hydroxylation (+32 Da mass shift) at different proline residues, between the spiked-in heavy isotope-labelled synthetic peptide and the peptide in the IP sample. (D) The MS/MS spectrum of the sequence KPLLSPIPELPEVPEM(O)TP(OH)SIPSIRR from the spiked-in heavy isotope-labelled synthetic peptide (scan no. 21243) and the peptide from the IP sample (scan no. 25891). The intensity behaviour of (E) HyPro diagnostic ion (m/z at 86.060) and (F) leucine/isoleucine (L/I) immonium ion (m/z at 86.097) under different NCE settings, using the synthetic hydroxylated peptide KPLLSPIPELPEVPEMTP(604)SIPSIRR and KPLLSPIPELPEVPEMTPSIP(607)SIRR (m/z at 704.15). All ions were extracted under ±10 ppm mass tolerance.
Figure 8 with 1 supplement
Mass spectrometry (MS) analysis of Repo-Man hydroxylation at P604.
(A) Diagram of proline hydroxylation sites analysis in asynchronous HeLa and HEK293 cells using SILAC (stable isotope labelling of amino acids in cell culture). Cells were grown in SILAC media for 6 passages and 24 hr before harvesting cells were treated with FG-4592 50 µM or DMSO. DMSO-treated cells (control) were light labelled, and FG-4592-treated cells are heavy labelled. (B) MS/MS spectra of endogenous Repo-Man peptide (KPLLSPIPELPEVPEMTPSIPSIRR), where matched fragment ions (b/y) were marked. Both the peptide precursor ion (m/z 708.15) and its corresponding fragment ions (y8 and y10) confirmed the identification of KPLLSPIPELPEVPEM(602-Ox)TP(604-OH)SIPSIRR. The diagnostic ion (m/z 86.06) of hydroxylated proline was also shown as ‘P-OH’. (C) Quantification analysis of P604 hydroxylation in Repo-Man in both HeLa and HEK293 cells, under the treatment of either DMSO or FG-4592. The bar chart was based on the intensity of the peptide containing P604 hydroxylation. (D) Quantification analysis of HIF1α in both HeLa and HEK293 cells, where the protein raw intensity was used for the bar chart since there’s no HIF1α detected in DMSO-treated cells. (E) Quantification analysis of Repo-Man (CDCA2) and Histone H4 in both HeLa and HEK293 cells, where H/L represents the heavy to light ratio using SILAC quantification.
Figure 8—figure supplement 1
FG4592 induces proline hydorxylation in HIF1α.
(A) Western blot analysis of HIF1α from HEK293 cell lysates treated with different concentrations and times of FG-4592 as indicated in the figure. Actin was used as a loading control. MG-132 was added for 3 hr before cells were harvested. (B) Sequence alignment of Repo-Man peptide region (covering P604) across different species is displayed.
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Figure 8—figure supplement 1—source data 1
PDF file containing original western blots for Figure 8—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108128/elife-108128-fig8-figsupp1-data1-v1.zip
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Figure 8—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/108128/elife-108128-fig8-figsupp1-data2-v1.zip
Figure 9
In vitro hydroxylation of Repo-Man synthetic peptide by recombinant PHD1.
The reactions were performed in parallel using Repo-Man (KPLLSPIPELPEVPEM(602-OX)TPSIPSIRR) and HIF1α (LDLEMLAPYIPMDDD) synthetic peptides as substrates in combination with (+) or without (-) recombinant PHD1. (A) MS/MS spectra of the hydroxylated peptide KPLLSPIPELPEVPEM(602-OX)TP(604-OH)SIPSIRR from Repo-Man. The mirror image shows the fragment ions matching results between the experimental hydroxylated peptide (top) and its synthetic standard peptide (bottom), where matched b/y ions were highlighted. (B) Detailed MS/MS spectra of the hydroxylated synthetic peptide KPLLSPIPELPEVPEM(602-OX)TP(604-OH)SIPSIRR from Repo-Man. The mirror image shows the fragment ions matching results of the synthetic peptide incubated with or without PHD1. The y8 ion represents the fragment of [P(604-OH)SIP(607)SIRR], while the y5 ion represents the fragment of [P(607)SIRR], which confirmed the hydroxylation takes place at P604 only when incubation with PHD1. (C) Dot blot analysis of the in vitro hydroxylation of HIF1α peptide, with and without recombinant PHD1 (upper dots). Titration of the OH-564 HIF1α peptide as a control of the primary antibody. Membrane was incubated with anti-OH-564 HIF1α and developed using far-red secondary antibody in typhoon. (D) Liquid chromatography-mass spectrometry (LC-MS) analysis of in vitro hydroxylation of HIF1α peptide (LDLEMLAPYIPMDDD). The left panel shows the retention time difference in LC separation profile of HIF1α synthetic peptide with or without PHD1, compared to the HIF1α hydroxylated (P546-OH) synthetic peptide. The mirror image in the right panel shows the MS/MS fragment ions matching results between the experimental hydroxylated HIF1α peptide (top) and its synthetic standard peptide (bottom), where matched b/y ions were highlighted.
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Figure 9—source data 1
PDF file containing original dot blots for Figure 9, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108128/elife-108128-fig9-data1-v1.zip
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Figure 9—source data 2
Original files for dot blot analysis displayed in Figure 9.
- https://cdn.elifesciences.org/articles/108128/elife-108128-fig9-data2-v1.zip
Figure 10
A flowchart summary of our workflow to identify proline hydroxylation sites.
Additional files
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Supplementary file 1
All hydroxylated proline sites of HEK293 and RCC4 dataset.
- https://cdn.elifesciences.org/articles/108128/elife-108128-supp1-v1.xlsx
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Supplementary file 2
High-confident hydroxylated proline sites of HEK293 and RCC4 dataset.
Score ≥ 40 and localisation prob>0.5. Diagnostic peak of HyPro highlighted.
- https://cdn.elifesciences.org/articles/108128/elife-108128-supp2-v1.xlsx
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Supplementary file 3
High-confident hydroxylated proline sites of HEK293 and RCC4 with the removal of M.
Score ≥ 40 and localisation prob>0.5.
- https://cdn.elifesciences.org/articles/108128/elife-108128-supp3-v1.xlsx
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Supplementary file 4
High-confident hydroxylated proline sites of HEK293 and RCC4 (FG inhibits), and the overlapping sites from both datasets.
Score ≥ 40 and localisation prob>0.5.
- https://cdn.elifesciences.org/articles/108128/elife-108128-supp4-v1.xlsx
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Supplementary file 5
Reactome pathway results of proline-hydroxylated proteins from HEK293 and RCC4 dataset, and the overlapping hydroxylated proteins from both datasets.
- https://cdn.elifesciences.org/articles/108128/elife-108128-supp5-v1.xlsx
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Supplementary file 6
PRM inclusion list of the target hydroxylated peptides from Repo-Man, HIF1α, CEP192, and PKM2.
- https://cdn.elifesciences.org/articles/108128/elife-108128-supp6-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108128/elife-108128-mdarchecklist1-v1.docx
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Systematic characterisation of site-specific proline hydroxylation using hydrophilic interaction chromatography and mass spectrometry
eLife 14:RP108128.
https://doi.org/10.7554/eLife.108128.3
